1. Volume 33, Issue 2, Pages 217-228.e4 (February 2018)
c-RAF Ablation Induces Regression of Advanced Kras/Trp53 Mutant Lung Adenocarcinomas by a Mechanism Independent of MAPK Signaling 
Manuel Sanclemente, Sarah Francoz, Laura Esteban-Burgos, Emilie Bousquet-Mur, Magdolna Djurec, Pedro P. Lopez-Casas, Manuel Hidalgo, Carmen Guerra, Matthias Drosten, Monica Musteanu, Mariano Barbacid 
Cancer Cell 
Volume 33, Issue 2, Pages e4 (February 2018)
DOI: /j.ccell
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2. Cancer Cell 2018 33, 217-228.e4DOI: (10.1016/j.ccell.2017.12.014)
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 1
c-RAF Ablation Reduces Tumor Burden in KrasG12V-Driven Lung Tumors
(A) Western blot analysis of c-RAF and B-RAF expression in lysates derived from five independent tumors obtained from Kras+/FSFG12V;RERTert/ert, Kras+/FSFG12V;RERTert/ert;c-RafL/L and Kras+/FSFG12V;RERTert/ert;BrafL/L mice exposed for 4 months to a TMX diet. GAPDH was used as the loading control. Migration of the above proteins is indicated by arrowheads.
(B) CT scan axial projection images and 3D reconstructions of lungs of representative Kras+/FSFG12V;RERTert/ert mice carrying the indicated c-Raf and Braf alleles before and after 4 months of TMX exposure. Tumors are indicated by arrowheads. H, heart.
(C) Quantification of average tumor volume variation (indicated as percent change) during the TMX exposure for the indicated time. Tumors were measured by CT scans. Kras+/FSFG12V;RERTert/ert (n = 25) (open circles); Kras+/FSFG12V;RERTert/ert;BrafL/L (n = 16) (solid gray circles); Kras+/FSFG12V;RERTert/ert;c-RafL/L (n = 22) (solid black circles) and Kras+/FSFG12V;RERTert/ert;c-RafL/L;BrafL/L (n = 8) (solid gray triangles) mice. Error bars indicate mean ± SEM.
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4. Figure 2
c-RAF Ablation Decreases Glucose Uptake in KrasG12V-Driven Lung Tumors
(A) PET/CT scan axial projections images and 3D reconstructions to assess metabolic activity ([18F]-FDG uptake) of representative lungs from Kras+/FSFG12V;RERTert/ert, Kras+/FSFG12V;RERTert/ert;c-RafL/L and Kras+/FSFG12V;RERTert/ert;c-RafL/L;BrafL/L mice before and after 2 months of TMX exposure. Tumors are indicated by arrowheads. H, heart. Color-scale bar represents SUV of PET.
(B) Quantification, indicated as percent change, of the number of tumors positive for [18F]-FDG uptake per mouse in Kras+/FSFG12V;RERTert/ert (n = 14) (open bar), Kras+/FSFG12V;RERTert/ert;c-RafL/L (n = 19) (solid black bar), and Kras+/FSFG12V;RERTert/ert;c-RafL/L;BrafL/L (n = 17) (solid gray bar) mice after 2 months of TMX exposure.
(C) Quantification, indicated as percent change, of the SUV levels of [18F]-FDG uptake per tumor, measured by PET scan, in Kras+/FSFG12V;RERTert/ert (n = 14 mice/30 tumors) (open circles), Kras+/FSFG12V;RERTert/ert;c-RafL/L (n = 19 mice/46 tumors) (solid black circles), and Kras+/FSFG12V;RERTert/ert;c-RafL/L;BrafL/L (n = 17 mice/37 tumors) (solid gray circles) animals after 2 months of TMX exposure.
Error bars indicate mean ± SEM. p values were calculated using the unpaired Student's t test. ∗∗p < 0.01 or ∗∗∗p < n.s., not significant.
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5. Figure 3
c-RAF Is an Effective Therapeutic Target for Advanced KrasG12V/Trp53 Mutant Tumors
(A) (Left) Waterfall plot representing the percent change in tumor volume of individual tumors from Kras+/FSFG12V;RERTert/ert mice (n = 25 mice/35 tumors) exposed to TMX for 4 months. (Right) Waterfall plot representing the percent change in tumor volume of individual tumors present in Kras+/FSFG12V;RERTert/ert;c-RafL/L mice (n = 22 mice/42 tumors) exposed to TMX for 4 months. The percent change in tumor volume was calculated by CT measurements performed for each individual tumor at the beginning and end of the 4-month-long trial.
(B) Waterfall plot representing the change in tumor volume of individual CT+ tumors present in Kras+/FSFG12V;Trp53F/F;hUBC-CreERT2+/T;c-Raf+/+ mice (n = 20 mice/54 tumors) exposed to TMX for 2 months. (Right) Waterfall plot representing the change in tumor volume of individual CT+ tumors present in Kras+/FSFG12V;Trp53F/F;hUBC-CreERT2+/T;c-RafL/L mice (n = 27 mice/94 tumors) exposed to TMX for 2 months. The percent change in tumor volumes were calculated for each individual tumor based on CT scans performed at the beginning and at the end of the 2-month-long trial.
(C) Table resuming the numbers of regressing and de novo detected tumors in the upper mentioned mice after exposure to TMX.
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6. Figure 4
MAPK and PI3K Signaling in KrasG12V/Trp53 Mutant Tumors after c-RAF Ablation
(A) Western blot analysis of K-RAS, H/N-RAS, B-RAF, c-RAF, phospho-MEK (pMEK), MEK1, phospho-ERK (pERK), ERK1/2, phospho-AKT (pAKT), and AKT expression in lysates derived from individual tumors (C1 to C4) of control Kras+/FSFG12V;Trp53F/F;hUBC-CreERT2+/T;c-Raf+/+ mice and of Kras+/FSFG12V;Trp53F/F; hUBC-CreERT2+/T;c-RafL/L mice exposed to TMX for 2 months. Progressor (P1-P4) and regressor (R1-R8) tumors analyzed are indicated. GAPDH was used as loading control. Migration of the above proteins is indicated by arrowheads.
(B) Waterfall plot representing the change in tumor volume of individual tumors of Kras+/FSFG12V;Trp53F/F;hUBC-CreERT2+/T;c-RafL/L mice previously shown in Figure 3. Tumors used for western blot analysis are indicated in red. Tumors used for phospho-ERK IHC analysis are indicated in blue. The scale used to represent tumor reduction is magnified 2-fold for better visualization.
(C) Phospho-ERK staining of representative sections obtained from the indicated tumors obtained from Kras+/FSFG12V;Trp53F/F; hUBC-CreERT2+/T;c-RafL/L mice treated with TMX for 2 months. Scale bars, 1 mm (R9 to R11 tumors) and 0.2 mm (P5, R12 to R15 tumors).
(D) Phospho-ERK IHC staining of representative tumor sections from tumors (C5 to C10) obtained from Kras+/FSFG12V;Trp53F/F;hUBC-CreERT2+/T;c-Raf+/+ mice exposed to TMX for 2 months. IHC of the sections depicted in (C) and (D) was carried out in parallel. Scale bar, 2 mm.
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7. Figure 5
c-RAF Ablation Induces Tumor Differentiation and Apoptosis through MAPK Independent Mechanisms
(A) (Left) Phospho-Cofilin staining and (right) quantification of phospho-Cofilin+ cells in representative sections of paraffin-embedded tumors of Kras+/FSFG12V;Trp53F/F;hUBC-CreERT2+/T;c-Raf+/+ (+/+, open bar) and Kras+/FSFG12V;Trp53F/F;hUBC-CreERT2+/T;c-RafL/L (L/L, solid bar) mice (n = 6 per genotype) exposed to TMX for 2 months. Scale bar, 0.05 mm.
(B) (Left) Cleaved Caspase 3 staining and (right) quantification of cleaved Caspase 3+ cells in representative sections of paraffin-embedded tumors of Kras+/FSFG12V;Trp53F/F;hUBC-CreERT2+/T;c-Raf+/+ (+/+, open bar) and Kras+/FSFG12V;Trp53F/F;hUBC-CreERT2+/T;c-RafL/L (L/L, solid bar) mice (n = 6 per genotype) exposed to TMX for 2 months. Cleaved Caspase 3+ cells are indicated by arrowheads. Scale bar, 0.05 mm.
(C) Western blot analysis of B-RAF, c-RAF, Caspase 3, cleaved Caspase 3, phospho-MEK, MEK1, phospho-ERK and ERK expression in lysates from KrasG12V;Trp53−/−;hUBC-CreERT2+/T;c-RafL/L cells maintained in 4OHT-containing media. Samples were harvested at the indicated times in hr. Vinculin was used as loading control. Migration of the above proteins is indicated by arrowheads. Three independent tumor cell lines from three different animals were evaluated.
Error bars indicate mean ± SEM. p values were calculated using the unpaired Student's t test. ∗∗∗p <
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8. Figure 6
c-RAF Expression Is Essential for Proliferation of Patient-Derived Xenograft Tumors
(A) Cell proliferation assay of a PDX-derived cell line (PDX-dc1 harboring K-RASG12C mutation) expressing scrambled shRNA (Scramb.) (solid circles) and shRNA against c-RAF (c-RAF) (open circles).
(B) Quantification of tumor number per mouse after orthotopic implantation of PDX-dc1 expressing scrambled shRNA (solid bar) and shRNA against c-RAF (open bar) in nude mice. Animals were analyzed at 5 weeks after the implantation. n = 10 mice/condition.
(C) Quantification of tumor volume after orthotopic implantation of PDX-dc1 expressing scrambled shRNA (solid bar) and shRNA against c-RAF (open bar) in nude mice. Animals were analyzed at 5 weeks after the implantation. n = 10 mice/condition.
(D) Cell proliferation assay of a PDX-derived cell line (PDX-dc2 harboring K-RASG12C mutation) expressing scrambled shRNA (Scramb.) (solid circles) and shRNA against c-RAF (c-RAF) (open circles).
(E) Quantification of tumor number per mouse after orthotopic implantation of PDX-dc2 expressing scrambled shRNA (solid bar) and shRNA against c-RAF (open bar) in nude mice. Animals were analyzed at 3 weeks after the implantation. n = 7 mice/condition.
(F) Quantification of tumor volume after orthotopic implantation of PDX-dc1 expressing scrambled shRNA (solid bar) and shRNA against c-RAF (open bar) in nude mice. Animals were analyzed at 3 weeks after the implantation. n = 7 mice/condition.
(G) (Left) Quantification of colonies forming in PDX-dc1 cells, PDX-dc1 cells expressing lentiviral pLVX control particles (empty vector) or expressing the wild-type murine c-Raf (Murine c-Raf) (as indicated). All cells indicated are expressing a scrambled shRNA. (Right) Quantification of colonies forming in PDX-dc1 cells, PDX-dc1 cells expressing lentiviral pLVX control particles (empty vector) or expressing the wild-type murine c-Raf (Murine c-Raf) (as indicated). All cells indicated are expressing a shRNA against human c-RAF.
Error bars indicate mean ± SEM. p values were calculated using the unpaired Student's t test. ∗∗p < 0.01 or ∗∗∗p < n.s., not significant.
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